Bioprinted Alginate Viability

Introduction

As a naturally occurring polysaccharide commonly derived from algae or seaweed, sodium alginate’s abundance and low cost make it a popular biomaterial for cellular encapsulation and bioprinting (1). Sodium alginate forms a hydrogel through a sodium-calcium ion exchange. As this crosslinking process occurs very quickly, sodium alginate can be difficult to bioprint on its own. Many studies combine this reagent with other materials to create a hybrid bioink or use complex multi-crosslinking steps to create 3D geometries (2-4). Here, we present a simplified process for printing sodium alginate with the use of Pluronic F127 as a temporary support. However, the FRESH method of bioprinting is suggested for the best resolution and for fabrication of more complex 3D structures. This process creates viable 3D structures of cell-laden alginate with repeatable, accurate geometries.

Results

Thin Film Viability

Bioprinted viability was first tested by comparing bioprinted thin films to pipetted thin films. Both 2% and 5% bioprinted thin films had over 85% viability compared to non-bioprinted controls. No statistical differences were found between 2% and 5% groups at each time point.

Figure 1: Live/Dead Images (left) of 2% and 5% bioprinted alginate thin films after 7 days of culture (scale bars = 1 mm). The data was quantified using Image J and viability data for 2% and 5% alginate thin films, normalized to pipetted thin film controls, is shown on the right.

Bioprinted Ring Viability

Figure 2: Live/Dead confocal images of pipetted thin film (left), bioprinted thin film (middle) and bioprinted ring (right) after 7 days of culture.

Ring structures designed with a 300 μm thickness, 400 μm height and an outer diameter of 2.3 mm were printed with 2% cell-laden alginate. Viability of these bioprinted rings were compared to bioprinted and pipetted thin films (figure 2). Qualitative analysis of live/dead imaging suggests similar viability in the bioprinted ring structures as the bioprinted and pipetted thin films. Line width averages, analyzed with ImageJ software, were 360 ± 80 μm after 1 day of culture.

Conclusions

These results demonstrate the ability of sodium alginate, calcium chloride and Pluronic F127 from Allevi to support viable cultures and create consistent, high resolution 3D geometries when used with an Allevi 1. When used together, these reagents can create cell-laden structures with specific, repeatible 3D geometries. Further, bioprinted thin films and pipetted thin films created in this study can be used as 3D controls for future experiments with these materials.

Thin Film Fabrication

HNDFs were mixed with either 2% or 5% (w/v) alginate at a 5 million/ml concentration. Pipetted thin films were created by pipetting 10 μl of solution on transwell plates then submerging the samples in 10 mM Calcium Chloride for 10 – 15 minutes.

2% Bioprinted thin films, printed with this gcode were sliced according to the suggested print parameters below. 5% bioprinted thin films, printed with this gcode, were also printed using the suggested print parameters. Volume of extruded bioprinted material was estimated with the volume test. Extrusion time in the printed gcode was set for extrusion of approximately 10 μl. All samples were crosslinked for 10-15 minutes in calcium chloride, then washed three times with phosphate buffered saline (PBS). All samples were bioprinted with an Allevi 2.

Bioprinted Ring Fabrication

First, CAD files of alginate rings and supporting pluronic F127 structure were created with solidworks. Then, these .stl files were loaded into repetier host and sliced with the suggested print parameters. 5 ml of Pluronic F127 was loaded into extruder 1 and 2 ml of 2% alginate with a 5 million/ml HNDF concentration was loaded into extruder 2. 30 gauge needles were used for both materials.

Rings were printed into transwell plates, then submerged in 10 mM calcium chloride first at room temperature for 5-10 minutes then on ice for another 5 minutes. After crosslinking, samples were washed with phosphate buffered saline (PBS) three times before adding media. All samples were bioprinted with an Allevi 2.

Printing Parameters

Extruder

Speed (mm/s)

Layer Height (mm)

Nozzle Diam (mm)

Gauge

Pressure

Print Temp(C)

Volume Fill (ml)

Alginate

6.0

0.1

0.1

30 untapered

7.5

25

3-5

Sample Analyses

To assess cell viability, a LIVE/DEAD kit (Life Technologies) was used. Images were taken on a Nikon TE300 Inverted Fluorescent Microscope or a Leica TCS SP5 II Scanning Laser Confocal Microscope. Statistical analysis was performed using an Analysis of Variance (ANOVA) test to determine if differences were present amongst treatment groups. If differences were determined from the ANOVA, a post hoc Tukey’s multiple comparison test was used to determine statistical differences between groups tested. A confidence level of 95% (α=0.05) was used for all analyses. Error bars on graphs represent the standard deviation from the mean.

Supplementary Information

After this study, sodium alginate was tested with the FRESH method. This method was able to achieve significantly better resolution and is suggested for optimal bioprinting results with alginate. Check out our FRESH kits here.

Figure 3: Bioprinted Alginate with the FRESH method is able to achieve a resolution of 0.15 +/- 0.03 mm.